Microscopes that allow a changeover between evanescent illumination and point-like scanning illumination of an object are known from the existing art.
WO 2005/031428 A1 discloses a microscope with evanescent sample illumination, which additionally comprises an optical apparatus for manipulating a sample. Here a laser serves to generate an illuminating light ray bundle that is focused into the back pupil plane of the microscope objective. A beam deflection device that encompasses a gimbal-mounted rotating mirror serves to adjust the lateral distance of the focus of this laser light ray bundle with respect to the optical axis of the objective. When the distance of the focus with respect to the optical axis of the objective is sufficient, total reflection occurs at the boundary surface between the sample holder (for example, a cover slip) and the sample. The evanescent field produced as a result penetrates only into the boundary surface of the sample and decreases exponentially with distance from that boundary surface. Fluorophores present in the boundary surface of the sample are thus excited, while background fluorescence can be considerably reduced. Image contrast is thereby improved. This method is referred to generally as total internal reflection fluorescence microscopy (TIRFM).
The apparatus for manipulating a sample encompasses, for example, a multi-line laser from whose emission spectrum the components having the desired wavelengths are selectable using an acousto-optical tunable filter (AOTF). The position of the manipulating light ray bundle that is focused into the sample is adjusted with a further beam deflection device.
In an embodiment, in order to change over between manipulation illumination and TIRF illumination, a hinged mirror is pivoted into the TIRF illuminating beam path so that the TIRF illuminating beam path strikes the non-reflective back side of the hinged mirror and is prevented from propagating further. In its pivoted-in position the hinged mirror is arranged in such a way that the manipulating light ray bundle is reflected at the hinged mirror and thereby coupled into the illuminating beam path. In another embodiment a single laser light source having selectable wavelengths is used both for TIRF illumination and for manipulation illumination. In this case a switchover between the two illumination modes is effected by simply introducing an adaptation optic into the illuminating beam path. The effect of this adaptation optic is that for TIRF illumination, the laser ray bundle is focused into the back pupil plane of the microscope objective. Without the adaptation optic, conversely, the laser ray bundle is focused into the sample. The manipulation light ray bundle is, as a rule, a laser scanning beam for (confocal) scanning microscopy.
In this document, optical components (hinged mirror or adaptation optic) are consequently introduced into or removed from the illuminating beam path in order to switch over between TIRF illumination and manipulation illumination.
A confocal scanning microscope generally encompasses a light source, a focusing optic with which the light of the source is focused onto an aperture (called the “excitation pinhole”), a beam splitter, a beam deflection device for beam control, a microscope optic, a detection pinhole, and detectors for detecting the detected or fluorescent light emitted from the sample. The illuminating light is coupled in via a beam splitter. The fluorescent or detected light emitted from the object travels via the beam deflection device back to the beam splitter, passes through it, and is then focused onto the detection pinhole behind which the detectors are located. This detection arrangement is called a “descan” arrangement. Detected light that does not derive directly from the focus region does not pass through the detection pinhole, so what is obtained is a point datum that, by sequential scanning of the object with the focus of the illuminating light ray bundle, can be assembled into a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers in different focal planes.
The subject matter of U.S. Pat. No. 7,187,494 B2 is a microscope having a laser light source, the microscope comprising a deflection unit for decentering the laser beam out of the optical axis, and an objective through which the laser beam passes. In an exemplifying embodiment a so-called collector lens arrangement, which is arranged introducibly into and removably from the illuminating beam path, is proposed for switching over between scanning microscopy and TIRF microscopy. When the collector lens arrangement is in the illuminating beam path, the laser light beam is focused decenteredly into the pupil of the objective (TIRF illumination), while when the collector lens arrangement is removed from the illuminating beam path the laser beam is focused onto the sample and scans it.
With this microscope it has proven to be disadvantageous that the switchover between TIRF microscopy and scanning microscopy is too slow for many applications, and that the alignment outlay for the elements introducible into the illuminating beam path is very high if the intention is to achieve precise beam guidance in both microscopy modes.
WO 2007/020251 A1 discloses a microscope for TIRF microscopy in which laser light of a laser light source is directed firstly via a hole in a mirror and then via a displacement unit toward a microscope objective. For the laser beam, the hole on the non-reflective side of the mirror presents itself as an ordinary aperture opening whose diameter corresponds to the diameter of the illuminating light ray at the location of the mirror. The reflective surface is located on the other side of the mirror. By way of the displacement unit, the laser beam acquires its decentering required for TIRF illumination. As a result, it can be incident at a critical angle onto the boundary surface between the sample carrier and object. For the transition from a cover slip having a refractive index n1=1.518 to water having a refractive index n2=1.33, for example, this critical angle has a value of 61° (the total reflection angle).
In an embodiment the laser beam is guided rotationally symmetrically with respect to the main axis of the object, by means of the displacement unit, in such a way that an evanescent field is generated in the object as a result of total reflection. The total reflection angle required for this can only be calculated if the refractive index of the sample is known. Often, however, different samples are involved, usually having unknown refractive indices. In order nevertheless to allow an empirical indication of the total reflection angle, i.e. to allow evanescent illumination to be established automatically, this document proposes detecting the reflected light that occurs before the critical total reflection angle is reached. This light travels through the microscope objective back to the displacement unit and from there onto the reflective surface of the mirror arranged in the illuminating beam path. From there the reflected light is reflected into a detector. The proportion of reflected light decreases drastically when the total reflection angle is reached, since the laser beam is now totally reflected in the sample carrier at the interface with the object, and excites the evanescent field. The position of total reflection, i.e. the degree of deflection by the displacement unit necessary therefor, can be determined from this transition.
In modern microscopes, especially inverted ones, investigation of living systems such as cells requires a variety of illumination methods for a single sample. For this, as a general rule, lasers having different wavelengths are switched in temporal succession at very short time intervals in order to excite or manipulate the sample and for subsequent measurement and evaluation. With the systems described above for TIRF microscopy and laser scanning microscopy, mirror elements and/or lens elements must be mechanically introduced into or removed from the illuminating beam path. When individual illumination modules are coupled together with the aid of such movable optical elements, each module has its specific laser light illumination system, and these must often be laboriously synchronized with one another for the experiment. Mechanical introduction and removal of the optical elements has a negative effect on the desired short time intervals between excitation/manipulation and measurement/evaluation. In addition, mechanical switching and shifting arrangements of this kind having the accuracy necessary for the application are mechanically complex and correspondingly expensive.
In addition to the illumination methods recited above, mention will also be made hereinafter of fluorescence recovery after photobleaching (FRAP) and Förster resonance energy transfer (FRET). In FRAP, a dye in the sample is destroyed (bleached) with X-Y precision at high intensities, and adjacent compartments are then excited via suitable illumination (for example TIRF illumination). If the bleached and excited compartments are connected, dye can travel by diffusion into the bleached regions. High switching rates are needed in order to detect this.
The FRET method uses fluorophores in which the donor is excited and the acceptor emits light as a result of resonance energy transfer. Precise spot laser scanning illumination at a suitable frequency allows the acceptors to be selectively destroyed (bleached), while the donors survive.
The intensity of the resonance energy transfer depends, inter alia, on the distance between donor and acceptor. If the distance is too great or if the acceptors are bleached, the donor itself then radiates the energy as a fluorescence emission. The distance necessary for resonance energy transfer is in the range from approximately 0.5 to 10 nm. A measurement of the radiation intensity of the donor dye in the absence and the presence of the acceptor dye allows inferences as to biochemical processes in the sample. For example, donor and acceptor dyes can be coupled to substances whose physical interaction is to be investigated. If an energy transfer that is measurable on the basis of the aforementioned radiation intensity takes place, the two substances are physically interacting. A lower switchover rate is required for the FRET method as compared with the FRAP method, but there are greater demands in terms of resolution for investigating biochemical processes in the cells being examined.
Multiphoton microscopy represents a further scanning imaging method. Here, nonlinear optical effects are generated with the aid of a focused high-energy laser beam. The construction and manner of operation of a multiphoton microscope are similar to those of a confocal laser scanning microscope. Whereas the latter achieves penetration depths from 50 to 80 μm (depending on the specimen), penetration depths of several hundred μm can be attained with multiphoton microscopy. This allows image production to extend into deeper tissue regions. In multiphoton fluorescence microscopy, two or more simultaneously incident photons are absorbed by an electron of a dye, and the transition into the ground state occurs with emission of a short-wave photon. The excitation photons, on the other hand, have a longer wavelength. With two-photon excitation the excitation wavelength is approximately twice the excitation wavelength normally used in fluorescence microscopy.
High photon densities are necessary in order to achieve simultaneous absorption of two or more photons, and can be furnished using high-intensity pulsed lasers. As in the case of a confocal laser scanning microscope, the laser beam is focused through the microscope's objective onto a point in the specimen. The focused laser beam is scanned over the specimen by means of a scanning unit. The emitted fluorescent radiation is directed to a detector via the objective and a dichroic beam splitter. The off-times between two laser pulses are sufficiently long that the energy introduced into the specimen can be re-emitted as fluorescent radiation in a fraction of that time. The detectors therefore measure the brightness of each image point, so that a complete image of the specimen can be assembled once the specimen has been scanned.
An advantage of multiphoton microscopy as compared with confocal laser scanning microscopy is the greater penetration depth. In two-photon microscopy, for example, the light used for excitation is infrared, which is scattered and absorbed much less than visible light in biological tissue. In contrast to confocal laser scanning microscopy, with multiphoton microscopy all of the fluorescence collected by the objective is used for the image that is to be created; a pinhole is therefore not needed in order to filter out light from other planes. This is because the light intensities outside the focal plane are not sufficient to cause multiphoton excitation of the fluorescent dye therein. It is therefore also not necessary to capture the fluorescent radiation via the scanning mirror (“non-descanned” detection). With a short-pulse laser and an integral detector, a system of this kind can be combined as a TIRF and multiphoton system.
The object of the present invention is to describe a microscope that makes possible, without mechanical introduction and removal of optical elements respectively into and from the illuminating beam path, a fast changeover between pointlike scanning illumination (or “image scanning”) and evanescent illumination (or “pupil scanning”). Hereinafter the illuminating beam path for pointlike scanning illumination will be called an “orthoscopic beam path,” and the illuminating beam path for evanescent illumination will be called a “conoscopic beam path.”